Bisphosphonate-based molecules as potential new antiparasitic drugs

Neglected tropical diseases such as Chagas disease and leishmaniasis affect millions of people around the world. Both diseases affect various parts of the globe and drugs traditionally used in therapy against these diseases have limitations, especially with regard to low efficacy and high toxicity. In this context, the class of bisphosphonate-based compounds has made significant advances regarding the chemical synthesis process as well as the pharmacological properties attributed to these compounds. Among this spectrum of pharmacological activity, bisphosphonate compounds with antiparasitic activity stand out, especially in the treatment of Chagas disease and leishmaniasis caused by Trypanosoma cruzi and Leishmania spp., respectively. Some bisphosphonate compounds can inhibit the mevalonate pathway, an essential metabolic pathway, by interfering with the synthesis of ergosterol, a sterol responsible for the growth and viability of these parasites. Therefore, this review aims to present the information about the importance of these compounds as antiparasitic agents and as potential new drugs to treat Chagas disease and leishmaniasis.publishersversionpublishe

Evidence for Sub-Haplogroup H5 of Mitochondrial DNA as a Risk Factor for Late Onset Alzheimer's Disease

BACKGROUND: Alzheimer's Disease (AD) is the most common neurodegenerative disease and the leading cause of dementia among senile subjects. It has been proposed that AD can be caused by defects in mitochondrial oxidative phosphorylation. Given the fundamental contribution of the mitochondrial genome (mtDNA) for the respiratory chain, there have been a number of studies investigating the association between mtDNA inherited variants and multifactorial diseases, however no general consensus has been reached yet on the correlation between mtDNA haplogroups and AD. METHODOLOGY/PRINCIPAL FINDINGS: We applied for the first time a high resolution analysis (sequencing of displacement loop and restriction analysis of specific markers in the coding region of mtDNA) to investigate the possible association between mtDNA-inherited sequence variation and AD in 936 AD patients and 776 cognitively assessed normal controls from central and northern Italy. Among over 40 mtDNA sub-haplogroups analysed, we found that sub-haplogroup H5 is a risk factor for AD (OR=1.85, 95% CI:1.04-3.23) in particular for females (OR=2.19, 95% CI:1.06-4.51) and independently from the APOE genotype. Multivariate logistic regression revealed an interaction between H5 and age. When the whole sample is considered, the H5a subgroup of molecules, harboring the 4336 transition in the tRNAGln gene, already associated to AD in early studies, was about threefold more represented in AD patients than in controls (2.0% vs 0.8%; p=0.031), and it might account for the increased frequency of H5 in AD patients (4.2% vs 2.3%). The complete re-sequencing of the 56 mtDNAs belonging to H5 revealed that AD patients showed a trend towards a higher number (p=0.052) of sporadic mutations in tRNA and rRNA genes when compared with controls. CONCLUSIONS: Our results indicate that high resolution analysis of inherited mtDNA sequence variation can help in identifying both ancient polymorphisms defining sub-haplogroups and the accumulation of sporadic mutations associated with complex traits such as AD

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 \ub1 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys

The Complete Genome Sequence of ‘Candidatus Liberibacter solanacearum’, the Bacterium Associated with Potato Zebra Chip Disease

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with ‘Candidatus Liberibacter solanacearum’, a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for ‘Ca. L. solanacearum’. Here we present the sequence of the 1.26 Mbp metagenome of ‘Ca. L. solanacearum’, based on DNA isolated from potato psyllids. The coding inventory of the ‘Ca. L. solanacearum’ genome was analyzed and compared to related Rhizobiaceae to better understand ‘Ca. L. solanacearum’ physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, ‘Ca. L. solanacearum’ is related to ‘Ca. L. asiaticus’, a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to ‘Ca. L. asiaticus’, ‘Ca. L. solanacearum’ probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles

A Redox Regulatory System Critical for Mycobacterial Survival in Macrophages and Biofilm Development

<div><p>Survival of <i>M</i>. <i>tuberculosis</i> in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel <i>r</i>edox <i>ho</i>meostati<i>c</i><i>s</i>ystem, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to <i>pknG</i>. Studies in <i>M</i>. <i>smegmatis</i> showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. <i>In vitro</i>, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities <i>in vivo</i> causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of <i>M</i>. <i>tuberculosis</i> in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.</p></div

Both <i>pknG</i> and its adjacent gene <i>renU</i> are each required for oxidative stress resistance.

<p>(<b>A</b>) Alignment of the <i>pknG</i> loci from <i>Mtb</i> and <i>M</i>. <i>smegmatis</i>. <i>renU</i> (previously annotated as <i>mutT3</i>) shares the same intergenic region with the operon encoding <i>pknG</i>. Bar, 1kb. (<b>B</b>) Both <i>pknG</i> and <i>renU</i> are each required for <i>M</i>. <i>smegmatis</i> resistance to H₂O₂ (left) and diamide (right). Wild type <i>M</i>. <i>smegmatis</i> (circles), <i>Ms</i>Δ<i>pknG</i> (triangles) and <i>Ms</i>Δ<i>renU</i> (squares) were grown in 7H9 medium. At the indicated times (arrows), 10mM H₂O₂ or 15mM diamide was added. Growth was estimated through optical absorbance at 600 nm (OD_600nm). Error bars represent standard deviation of biological triplicates. (<b>C</b>) <i>pknG</i> and <i>renU</i> are each required for <i>Mtb</i> resistance to H₂O₂ (left) and diamide (right). Wild type <i>Mtb</i> (circles or striped bars), <i>Mtb</i>Δ<i>pknG</i> (triangles or black filled bars) and <i>Mtb</i>Δ<i>renU</i> (squares or grey filled bars) of were grown in 7H9-OADC medium. At the indicated times (arrows), 20 mM H₂O₂ or 10 mM diamide was added. Growth was estimated through measuring optical absorbance at 600 nm (OD_600nm, top) or determining colony forming units (CFU, bottom) by serial dilution plating. Error bars represent standard deviation of biological triplicates.</p

Correlation of NADH and RHOCS, and role of L13 phosphorylation by PknG.

<p>(<b>A</b>) Induction of PknG expression in <i>M</i>. <i>smegmatis</i>. Western analysis was used to detect PknG expression following the exposure of wild type <i>M</i>. <i>smegmatis</i> cultures (OD₆₀₀ of 2) to various oxidative stimuli including NADH (upper) and FAD (lower) for 30 minutes. All chemicals were used at 10 mM except for bleomycin, which was used at 10 μg/ml. Samples were separated on SDS-PAGE, followed by immunodetection using an anti-PknG antibody or an anti-DivIVA antibody, as a control. Non-induced lysates from wild type <i>M</i>. <i>smegmatis</i> and <i>Ms</i>Δ<i>pknG</i> were used as controls. NADH uniquely induced expression of PknG. (<b>B</b>) Titration of the induced PknG expression by increasing NADH concentrations (0–30 mM) for 30 minutes, followed by Western analysis using anti-PknG antibody. (<b>C</b>) Time course of PknG expression (0–60 minutes) following cell exposure to 10 mM NADH. Detection of PknG was similar to (A) and (B). (<b>D</b>) Quantitation of cellular NADH (top), NAD⁺ (middle), and FAD (bottom) levels following oxidative stress induced by H₂O₂. <i>M</i>. <i>smegmatis</i> cells were exposed to 1 mM H₂O₂ for 1 hour. Bars show means with standard deviations from 3–6 biological repeats. *, p < 0.0001; ns, not significant relative to wild type <i>M</i>. <i>smegmatis</i>). (<b>E</b>) Effect of PknG-catalyzed phosphorylation of L13 on its association with RenU in the cytoplasm. Expression of PknG in <i>M</i>. <i>smegmatis</i> strains was induced by NADH. Cells were disintegrated by French Press, followed by ultracentrifugation to remove ribosomes. RenU.6H was added to the non-ribosomal fraction, followed by pull-down using Cobalt-agarose beads. The presence of L13 in the pulled down materials was detected by Western analysis using anti-L13 antibody. (<b>F</b>) Effect of L13(T11E), a phosphorylation-mimic form of L13, on <i>in vitro</i> NADH hydrolytic activity of RenU. Initial rates from a continuous fluorescence excitation assay were fit by nonlinear least squares to the Michaelis-Menten equation to determine K_m and V_max values for RenU. Reaction was performed at 37°C. Error bars represent standard deviations of triplicates. The extent of the uncatalyzed reaction was ~10% of the RenU catalyzed reaction. (<b>G</b>) Effect of L13(T11E) on the catalytic activity of RenU. In the presence of L13(T11E), a 20.6% increase in V_max was observed (p < 0.05x10^-3), whereas K_m, reflecting the binding affinity of RenU to NADH, was not affected by L13(T11E).</p